PROJECT SUMMARY The introduction of combined antiretroviral therapy has decreased the prevalence of HIV-associated dementia, but prevalence of milder HIV-associated neurocognitive disorders (HAND) has increased. The CNS is highly vulnerable to insult from HIV-1 via release of neurotoxic HIV proteins, inflammatory factors, and excitotoxins by infected astrocytes and microglia. Opiate use is often comorbid with HIV-1 infection and there is evidence for enhanced HAND symptomology in users, likely due to convergent cell signaling pathways resulting in increased neuronal dysfunction. Despite intense research the interactions between HIV-1 and opiates that lead to neuronal damage remain elusive, and most of this work is conducted in animal models. Since HIV-1 is a human-specific disease, it is paramount to validate and expand upon research findings in nonhuman models using human models. To this end, we have differentiated human neural progenitors to develop two primary human CNS culture models: i) a dissociated system containing glutamatergic and GABAergic neurons; and ii) a 3-dimensional brain aggregate (BrAgg) model that contains all major CNS cell types. Using non-invasive electrophysiological techniques including genetically encoded voltage and Ca2+ indicators (GEVI/GECI), and optogenetics we will examine the effects of infectious HIV-1, Tat, and gp120 morphine on primary human neuron function. By studying HIV-1 morphine effects on neuronal excitability we have uncovered a novel therapeutic target. K-Cl cotransporter 2 (KCC2) maintains low neuronal [Cl-]i necessary for ?-aminobutyric acid type A receptor (GABAAR)-mediated inhibition. Loss of KCC2 activity resulting in loss of GABAAR hyperpolarization, and even GABA-induced depolarization, has been implicated in a variety of neurological disorders. This project tests the hypothesis that HIV-1 and morphine-induced signaling converge to reduce neuronal KCC2 activity leading to electrophysiological and synaptic excitatory-inhibitory imbalance in primary human neurons. Our data have demonstrated significant loss of KCC2 and subsequent shift from hyper- to depolarization in response to GABA in HIV-1, Tat, and gp120 morphine-exposed human neurons. Pharmacological intervention to raise KCC2 levels has reversed these effects. Aim 1 will utilize GEVI and GECI to determine functional responses to infectious HIV-1, Tat, and gp120 (R5-, X4-, and dual-tropic) exposure on dissociated human neurons and examine modulation of KCC2 and relevant upstream regulators (e.g. NMDAR, CCR5, CXCR4, opioid receptors) to rescue noted deficits. Aim 2 will utilize stable BrAgg which contain all major CNS cell types and can be actively infected by HIV-1. Tools and measures described in Aim 1 will be used to examine neuronal function in a chronic, HIV-1-infected environment and elucidate differences between chronic, acute, and withdrawal morphine exposure paradigms. These studies use innovative models and techniques to determine effects of live HIV-1, Tat, and gp120 morphine on primary human neuron function and identify novel points of HIV-1 and morphine signaling convergence and targets for intervention to alleviate the symptoms of HAND comorbid opiate use.